Bryostatin-1 enhances barrier function in T84 epithelia through PKC-dependent regulation of tight junction proteins

Abstract

Protein kinase C (PKC) is known to regulate epithelial barrier function. However, the effect of specific PKC isozymes, and their mechanism of action, are largely unknown. We determined that the nonphorbol ester PKC agonist bryostatin-1 increased transepithelial electrical resistance (TER), a marker of barrier function, in confluent T84 epithelia. Bryostatin-1, which has been shown to selectively activate PKC-alpha, -epsilon, and -delta (34), was associated with a shift in the subcellular distribution of the tight junction proteins claudin-1 and ZO-2 from a detergent-soluble fraction into a detergent-insoluble fraction. Bryostatin-1 also led to the appearance of a higher-molecular-weight form of occludin previously shown to correspond to protein phosphorylation. These changes were attenuated by the conventional and novel PKC inhibitor Gö-6850 but not the conventional PKC inhibitor Gö-6976 or the PKC-delta inhibitor röttlerin, implicating a novel isozyme, likely PKC-epsilon. The results suggest that enhanced epithelial barrier function induced by bryostatin-1 involves a PKC-epsilon-dependent signaling pathway leading to recruitment of claudin-1 and ZO-2, and phosphorylation of occludin, into the tight junctional complex.

Role for TGF-β in Cyclosporine-Induced Modulation of Renal Epithelial Barrier Function

“…(i) Representative blot occludin in the maintenance of a functioning tight junction complex, and evidence suggests that it is the claudin proteins that play a pivotal role in the regulation of tight junctions. After exposure to CsA, increased expression of claudin-1 and claudin-3 was observed, both of which are implicated in the maintenance of tight junction integrity (32)(33)(34) No alterations in claudin-2 expression were detected after CsA treatment. Claudin-2 is associated with increased "leakiness" across epithelial cells.…”

Epidermal Growth Factor Receptor Activation Differentially Regulates Claudin Expression and Enhances Transepithelial Resistance in Madin-Darby Canine Kidney Cells

“…Claudin-1-deficient mice manifest a severely compromised epidermal barrier and die within 24 h of birth due to dehydration (62). Furthermore, bryostatin-1-induced tightness of the TJs correlates with enhanced membrane association of claudin-1 in T84 epithelial cells (48). Overexpression of claudin-4 has been shown to increase TER in MDCK II cells (49), and a recent study describing segmental distribution of various claudins in the mouse nephron showed that claudin-3 and -4 are expressed mainly in the "tight" epithelia of the kidney and that only claudin-1, -3, -4, and -8 are expressed in the collecting segment of the kidney nephron (60), the segment with the highest TER.…”

“…EGF Induces Intracellular Redistribution and Increases Protein Synthesis of Claudin-1-Overexpression of claudin-1 has been shown to increase TER and the number of TJ strands in MDCK II cells (46,47), and bryostatin-1-induced TJ tightness in T84 epithelial cells correlates with enhanced membrane association of claudin-1 (48). In this study, in contrast to claudin-2, EGF treatment induced significant increases in claudin-1 expression selectively in the detergent-insoluble fractions within 1 h after EGF treatment, and claudin-1 expression was 213 and 233% of the control values after 8 and 18 h of EGF treatment, respectively (Fig.…”

Acid modulates the squamous epithelial barrier function by modulating the localization of claudins in the superficial layers

“…This is consistent with the data from skin. 24 As the functional TJ proteins are insoluble in nonionic detergent, 10,12,29,30 in the present study, proteins from ALIcultured cells were divided into nonionic detergent (NP-40)-soluble and -insoluble fractions, and the level of claudin-1 and claudin-4 in each fraction then measured. Claudin-4 levels were lower in pH 2-stimulated cells compared with control conditions, in a time-dependent manner, suggesting that acid stimulation delocalized the claudin-4 from the TJs in the superficial layers.…”